In microscopy, an area of interest in a specimen to be imaged is often larger than can be displayed by taking a single image with the microscope. Thus scanning techniques are employed to image an entire desired area. In automated scanning, the specimen is moved under the objective lens of the microscope by an XY translation stage so that the microscope can scan across the desired area, with multiple images being collected and then aggregated or stitched to form a single larger image. This stitching can be accomplished using standard software techniques or by ensuring that images are taken at specific locations with very precise stage movement feedback such that, when a first image is taken, the stage moves exactly the distance equal to the width of the first image (and without movement in the height direction) and a second image is taken to them be joined at the common border. If precise enough, the left edge of first image will then exactly mate and compliment the right edge of for the second image.
It is often advantageous to align the camera pixels to a specific orientation relative to the specimen and then scan that specimen in a specific desired direction and in a manner that maintains the desired orientation. For example, components on silicon wafers (e.g., micro electronic devices or patterned films such as through photolithography) are often oriented in rows (x-direction) or columns (y-direction), and it is helpful to align the camera pixel rows parallel to a component row or align the camera pixel columns parallel to a component column to then accurately scan along a desired row or column while maintaining the parallel relationship there between.
In the current state of the art the camera pixel orientation is often manually aligned to the XY travel of the stage by visible observation, which, in light of the size scales typically involved, does not provide a suitable level of accuracy for many imaging needs. The chances of accurately aligning the pixel rows with the x-direction of the stage and the pixel columns with the y-direction of the stage are very low. After this likely inaccurate alignment, the specimen is rotated relative to the stage in an attempt to align the pixel rows of the image sensor with a desired x′-direction for scanning the specimen and/or to align the pixel columns with a desired y′-direction for scanning the specimen. That is, the specimen is rotated relative to the XY translation stage in order to position a desired x′ scanning direction of the specimen parallel to the x-direction movement of the stage and/or position a desired y′ scanning direction of the specimen parallel to the y-direction movement of the stage (i.e., the x′-direction and x-direction are intended to be the same and the y′-direction and y-direction are intended to be the same). The x-direction of the XY stage and the rows of pixels of the camera having been previously visually aligned (as are, axiomatically, the y-direction of the stage and the columns of pixels), the movement of the stage in the desired x-direction or desired y-direction maintains the desired alignment but only to the extent the manually alignment of the image sensor pixels to the XY travel of the stage is highly accurate and precise.
Returning to the patterned silicon wafer example, a desired x-direction might be a row of micro-circuits with this row being aligned parallel to the x-direction movement of the XY translation stage, and, thus parallel to the rows of pixels of the camera. The row of micro-circuits can thus be scanned simply by moving the XY translation stage in the x-direction, while the parallel relationship between the rows of pixel and the row of microcircuits is maintained, and the image sensor is not shifted in the y′-direction, such that accurate recording and stitching is facilitated.
Thus, accurate results depend upon highly accurate alignment of the camera pixels, the XY travel of the stage, and the desired x′ and/or y′ scanning directions of the specimen. This is difficult to achieve given normal tolerance in machining and errors inherent in mere visual observation alignment. If even slightly out of alignment, the image sensor will be shifted in the x′-direction and/or y′-direction to an unacceptable degree as the specimen is moved by the translation stage, thus frustrating the ease by which images can be analyzed and/or stitched together. Additionally, it is often desired that a specimen be analyzed with a minimal amount of handling of the specimen. Thus, there is a need in the art for new methods for aligning and scanning that do not rely on specimen movement and ensure accurate alignment between the image sensor and the specimen.